Lactic acid bacteria have long been used in a wide variety of industrial fermentation processes. They have generally-regarded-as-safe (“GRAS”) status, making them potentially useful organisms for the production of commercially important proteins. Indeed, several heterologous proteins, such as Interleukin-2, have been successfully produced in Lactococcus spp (Steidler et al., 1995). It is, however, undesirable that such genetically modified microorganisms survive and spread into the environment.
To avoid unintentional release of genetically modified microorganisms, special guidelines for safe handling and technical requirements for physical containment are used. Although this may be useful in industrial fermentations, the physical containment is generally considered as insufficient, and additional biological containment measures are taken to reduce the possibility of survival of the genetically modified microorganism in the environment.
Biological containment is extremely important in cases where physical containment is difficult or even inapplicable. This is, amongst others, the case in applications where genetically modified microorganisms are used as live vaccines or as a vehicle for delivery of therapeutic compounds. Such applications have been described, for example, in PCT. International Publication Number WO 97/14806, which discloses the delivery of biologically active peptides, such as cytokines, to a subject by recombinant noninvasive or nonpathogenic bacteria. Further, PCT International Publication Number WO 96/11277 describes the delivery of therapeutic compounds to an animal or human by administering a recombinant bacterium encoding a therapeutic protein. Steidler et al. (2000) describe the treatment of colitis by administration of a recombinant Lactococcus lactis secreting Interleukin-10. Such a delivery may indeed be extremely useful to treat a disease in an affected human or animal, but the recombinant bacterium may act as a harmful and pathogenic microorganism when it enters a nonaffected subject, and an efficient biological containment that avoids such unintentional spreading of the microorganism is needed.
Biological containment systems for host organisms may be passive and based on a strict requirement of the host for a specific growth factor or a nutrient that is not present or is present in low concentrations in the outside environment. Alternatively, it may be active and, based on so-called suicidal genetic elements in the host, wherein the host is killed in the outside environment by a cell-killing function, encoded by a gene that is under the control of a promoter only being expressed under specific environmental conditions.
Passive biological containment systems are well known in microorganisms such as Escherichia coli or Saccharomyces cerevisiae. Such E. coli strains are disclosed, for example, in U.S. Pat. No. 4,190,495. Also, PCT International Publication Number WO 95/10621 discloses lactic acid bacterial suppressor mutants and their use as means of containment in lactic acid bacteria, but in that case, the containment is on the plasmid level, rather than on the level of the host strain and it stabilizes the plasmid in the host strain, but does not provide containment for the genetically modified host strain itself.
Active suicidal systems have been described by several authors. Such systems consist of two elements: a lethal gene and a control sequence that switches on the expression of the lethal gene under nonpermissive conditions. For example, PCT International Publication Number WO 95/10614 discloses the use of a cytoplasmatically active truncated and/or mutated Staphylococcus aureus nuclease as a lethal gene. PCT International Publication Number WO 96/40947 discloses a recombinant bacterial system with environmentally limited viability, based on the expression of either an essential gene, expressed when the cell is in the permissive environment and not expressed or temporarily expressed when the cell is in the nonpermissive environment, and/or a lethal gene, wherein expression of the gene is lethal to the cell and the lethal gene is expressed when the cell is in the nonpermissive environment but not when the cell is in the permissive environment. PCT International Publication Number WO 99/58652 describes a biological containment system based on the relE cytotoxin. However, most systems have been elaborated for Escherichia coli (Tedkin et al., 1995; Knudsen et al., 1995; Schweder et al., 1995) or for Pseudomonas (Kaplan et al., 1999; Molino et al., 1998). Although several of the containment systems theoretically can by applied to lactic acid bacteria, no specific biological containment system for Lactococcus has been described that allows the usage of a self-containing and transformed Lactococcus to deliver prophylactic and/or therapeutic molecules in order to prevent and/or treat diseases.